: Phage Mu is an extraordinarily efficient transposon. Many fundamental insights into the mechanism of transposition have resulted from analysis of Mu recombination. Key aspects of this mechanism are common features used by diverse transposable elements that invade prokaryotic and eukaryotic organisms. The goal ol this project is to understand the mechanism of Mu transposition. The emphasis is on how the transposition proteins promote recombination and how the architecture of the protein-DNA complexes relates to mechanisms: of biological control. Mu transposase is a member of the structurally related transposase/retroviral integrase protein family. These proteins catalyze recombination as multimeric complexes bound to DNA. Key to understanding both mechanism and regulation is a clear picture of how DNA segments and regulatory proteins interact with the multimeric transposase. The first specific goal of this proposal is aimed toward understanding how the protein-DNA contacts between the transposase and the DNA at the old and new insertion sites are made, and how these contacts change as the recombination pathway progresses. Initial experiments test a model based on genetic analysis of Mu transposase and structural comparisons between the Mu transposase catalytic domain and the Tn5 transposase-DNA complex. I also propose experiments to identify molecular interactions between \lu transposase and its regulatory protein, MuB, and elucidate how these interactions enable Mu to regulate DNA target site choice. Similarly, studies designed to identify protein-DNA complexes and DNA intermediates used during the Mu non-replicative transposition pathway are also proposed. Finally, using biochemical and structural probes for protein conformational changes, I propose to test specific models describing how the Mu tranposaseL) NA complex is destabilized by the catalytic action of the ClpX chaperone. Transposition has a profound impact: on human health. Antibiotic resistance genes are spread by transposable elements moving throughout bacterial populations. Furthermore, retroviruses, including HIV, integrate into the host chromosome via a mechanism nearly identical to transposition. It is now also clear that DNA transposons have provided the substrate for evolution of numerous human genes, including those for the Rag recombinase that assembles antibody and T-cell receptor gene segments. Thus, understanding the molecular mechanism of transposition is essential for a thorough understanding of the molecular processes contributing to human diseases.